Fluorometry is an important quick and nondestructive analytical chemistry technique. Fluorometry is used to acquire both qualitative and quantitative data, and is of great interest for use in clinical chemistry and medical diagnostics as a means for measuring unknowns such as the pH and partial pressure of blood gasses and blood analytes.
In general, fluorometric analysis involves shining an energetic light onto a sample and stimulating the immediate re-emission or fluorescence of light of a particular frequency from the sample. The frequency of the light so fluoresced is characteristic of the particular sample component fluorescing. The frequency of the light shined onto the sample is usually chosen to be slightly higher than that of the frequency of the light characteristically fluoresced by the sample component desired to be measured. In other words, the fluoresced light has an energy less than or equal to that of the light source, since conservation of energy and the quantum nature of light dictate that the fluoresced photons cannot be more energetic than the excitation photons absorbed to produce the fluoresced photons.
The intensity of the fluoresced light is proportional to the quantity of the fluoresced sample. The fluorescence from the excited sample also has a finite and measurable lifetime. The fluorescent lifetime of a given material can be changed by the presence of an analyte such as oxygen and Ru(dpp).sub.3 and can be the basis of quantitative analysis. The motivation to use fluorescent lifetime measurements instead of intensity-based fluorescent measurements arises from the relative immunity of fluorescent lifetime measurements from many of the potential sources of error to which intensity measurements are prone. Examples of sources of error afflicting fluorescent intensity measurements include variations in the intensity of the light source or quantum efficiency of the detector, opacity or scattering characteristics of the sample medium, and geometrical differences between the source and the detector. By measuring the fluorescent lifetime instead of the fluorescent intensity, especially in biological samples, most or all of these sources of measurement error are minimized or eliminated.
There are two techniques commonly used to measure fluorescent lifetime: the pulse method and the harmonic modulation method. The pulse method involves measuring the lifetime of the fluoresced signal by fluorescing a sample with a pulsed source signal and measuring the pulse response of the fluorescent signal. The lifetime of the corresponding fluorescent pulse is measured in the time domain and an estimate of the lifetime is obtained by fitting a theoretical curve to the data.
The second technique for measuring fluorescent lifetime involves measuring the lifetime of the fluorescence in the frequency domain as a phase shift of the detected signal relative to the source signal. The relationship between the phase shift .phi. and the lifetime .tau. for a single lifetime fluorescent indicator expressed by: EQU tan .phi.=.omega..tau.,
where .omega. is the angular frequency of excitation of a known harmonic. By measuring .phi., the fluorescent lifetime .tau. can be calculated.
While a number of methods exist which allow the measurement of the phase of a fluoresced signal relative to a reference signal, all of the presently known methods require that the measured phase delay of the fluoresced signal be referenced to the measured phase delay of a fluorescent standard having a known lifetime, for example Rodamine B. This is necessary because all measurement systems have inherent phase delays arising from the measurement electronics. Without the reference standard, the phase measurement would incorporate the phase delay contribution from the electronics in the measurement of an unknown sample's phase delay, giving rise to potentially nontrivial errors. In addition to the referencing of a measured phase delay to a reference standard, some measurement systems, especially those using phase sensitive detection where the sample signal .SIGMA.A.sub.n sin(n.omega..tau.+.phi..sub.n) is multiplied by a reference signal B sin(.omega..tau.), require the subtraction of "artifacts" due to RF coupling or DC offsets from the measured signal. This typically requires the removal of the sample to ensure correct subtraction of these terms. Because the present methods require user intervention and a reference standard, the need arises for a method of measuring fluorescent lifetime that eliminates both user intervention and the requirement of a separate fluorescent standard. The present invention addresses this need.